The present mass screening method for the detection of gonorrhea is a bacteriological method. It requires two to seven days for completion because it necessitates waiting for growth of a colony of gonococcus organisms in an appropriate culture medium and confirmatory biochemical reactions by growth in a fermentation medium, such as shown in Table I. Moreover, the bacteriological method requires that a specimen of the gonorrhea caused discharge arrive at the testing laboratory with the fragile gonococcus organism still viable, a natural time limit of as little as two days. There is real need for improvement.
A serological method which would be capable of detecting antibodies in a blood sample would be highly desirable for use in a mass screening program to demonstrate that an individual may be currently suffering from gonorrhea or had been infected in the past. Individuals reacting positively could then be tested by the bacteriological method to determine if the infection is current. Several serological methods have been reported, but none is completely satisfactory. The primary reason for dissatisfaction has been the low sensitivity and the high number of false positive reactions.
A serological method has been developed which substantially alleviates the difficulties with known serological techniques, and is suitable for use in mass screening programs as an adjunct to the bacteriological method with substantial elimination of false positive reactions. This method is described and claimed in the above identified patent application.
This method is based upon the discovery that Neisseria gonorrhoeae (N.g.) organisms produce a species specific antigen which when present on the whole cell or in crude extracts is inactivated by heat and is trypsin sensitive. The antigen reacts specifically with antiobodies in patients sera which are produced as a result of an infection elicited by N.g. organisms. This interaction can be demonstrated by any of several techniques normally employed for these purposes. Most of them are discussed in the aforesaid application.
This antigen which has been called L-antigen, has been extracted as an L-antigen preparation or composition by a mild detergent procedure, and its characteristics described in an article by Chen N. C., Karmei K., Zucherman J., and Gaafar H. A., which was published in Infection and Immunity Vol. 18, p. 230-236. Another procedure which resulted in the preparation of an antigen immunologically indistinguishable from that prepared by the detergent procedure was described by Schraeder, Janet A., and Gaafar, H. A. published in Health Laboratory Science Vol. 15, p. 15-21.
The forms of L-antigen isolated by the described procedure show the following common characteristics:
1. Specie specificity PA1 2. The antigen mixture contains protein and carbohydrate. PA1 3. Heat lability in aqueous media. The ability to react, with specific antibodies present in infected patients' sera is reduced or eliminated by heating at 100.degree. C. for 1 hr. PA1 4. Stable in aqueous media at pH values of 3-11. PA1 5. Soluble in aqueous media at low concentration but insoluble in methanol, chloroform and acetone. PA1 6. Reacts with specific rabbit anti-human serum. PA1 1. Trypsin sensitive PA1 2. Resistant to dextranese, neuraminidase, DNA-ase, and RNA-ase. PA1 3. A molecular weight of antigenically active subunit of 37,000-40,000 as determined by SDS-PAGE. PA1 1. It stimulates the production of specific antibodies when used to immunize rabbits. The antiserum produced reacts with "L" antigen specifically irrespective of its stage of purity or method of extraction. This antiserum will produce only one precipitin line against "L" antigen preparations in immuno diffusion, countercurrent, or two dimensional immunoelectrophoresis. PA1 2. It is more resistant to heat. It retains activity even after boiling in water for 1 hr. In this application, this antigen preparation is referred to as heat resistant to differentiate it from the more heat labile forms previously described. PA1 3. stable when incubated with the following enzymes: PA1 4. inactivated when incubated with trypsin, PA1 5. molecular weight of active subunits of 37,000-40,000 as determined by SDS-polyacrylamide gel, PA1 6. isoelectric point of 4.+-.0.2, PA1 7. contains 2-3% organic phosphorus, PA1 8. soluble in aqueous media containing surface active agents, and insoluble in methanol, chloroform, and acetone, and PA1 9. reacts specifically with antibodies produced in rabbits against purified L-antigen, PA1 10. contains less than 1% carbohydrate, PA1 11. inactivated when incubated with trypsin. PA1 Heat labile L-antigen refers to the heat labile product isolated in accordance with the above-identified patent application. Besides heat lability, it differs from other L-antigen preparations by the presence of 1 10% of carbohydrate and other factors. Heat stable L-antigen refers to the product isolated in accordance with this invention. It differs from heat labile L-antigen in that it contains less than 1% carbohydrate, and, in fact, may be essentially carbohydrate free. Purified L-antigen refers to the product used to immunize rabbits to produce the anti-serum or antibody which reacts specifically with L-antigen. PA1 Order: Eubacteriales PA1 Family: Neissericeae PA1 Genus: Neisseria PA1 Species: Gonorrhoeae PA1 1. Forming a cell pellet of N.g. cells and extracting. PA1 2. Passing resulting extract over a molecular sieve column. It has been observed that the most useful sieves are those with an exclusion limit of at least 200,000. The L-antigen is always found in the void volume. PA1 3. Adding trichloracetic acid. PA1 4. Separating the precipitate. PA1 The antigen is diluted with saline and mixed with an aqueous suspension of fine charcoal particles by adding one volume of charcoal (2.5 mg/ml) to 8 volumes of antigen in a tube. The reagents are mechanically agitated using a Vortex mixer for 5 min. at room temperature and brought to 10 volumes with glycine buffered saline, pH 8.2 (73.1 g glycine, 50 g NaCL, 10 g bovine serum albumin, and 35 ml of 1.0 N sodium hydroxide per 1000 ml). PA1 The sera to be tested are diluted 1:10 in physiological saline and kept at 59.degree. C. for 30 min. The serum (0.05 ml) is placed in a circle printed on a plastic-surfaced card, and spread within the circle with a wooden toothpick. Sensitized charcoal (0.016 ml), prepared as above, is then added, and the card rotated for 8 min. at 180 rpm, hand-tilted, and left horizontal for 2 min. The results may be read under direct light using a 10X magnifying lens. The agglutination is fine, and any degree of agglutination may be considered positive.
The preparation described by Chen et al. has these additional characteristics:
On the other hand, the antigenic preparation of Schrader et al. had higher carbohydrate content, and was trypsin resistant.
These data confirmed that the physical characteristics of the partially purified L antigen are dependent on the method of extraction and purification and implies that these preparations contain other factors which affect the detectability of the L-antigen.
These antigenic preparations are useful for serological testing in accordance with procedures described in the aforementioned patent application such as indirect immunofluorescence, agglutination, enzyme linked immunoassay, radio-immunoassay, complement fixation tests, precipitation, and countercurrent immunoelectropheresis. The specificity of the tests is improved by prior heating of the patient serum to inactivate the naturally occurring antibodies which might react with the contaminating factors in the antigen preparation and/or by prior absorption of the serum with a sorbent made of N.meningitides cells as described in U.S. Pat. No. 4,029,756.